Abasic lesions are unable to form Watson-Crick hydrogen bonds with nucleotides. Nonetheless, polymerase and repair enzymes distinguish between various oxidized abasic lesions, as well as from nonoxidized abasic sites (AP). The C2-AP lesion is produced when DNA is exposed to gamma-radiolysis. Its effects on polymerases and repair enzymes are unknown. A recently reported method for the chemical synthesis of oligonucleotides containing C2-AP at a defined site was utilized for studying the activity of Klenow exo(-) and repair enzymes on templates containing the lesion. The C2-AP lesion has a similar effect on Klenow exo(-) as do AP and C4-AP sites. Deoxyadenosine is preferentially incorporated opposite C2-AP, but extension of the primer past the lesion is strongly blocked. C2-AP is incised less efficiently by exonuclease III and endonuclease IV than are other abasic lesions. Furthermore, although a Schiff base between C2-AP and endonuclease III can be chemically trapped, the location of the 3'-phosphate alpha with respect to the aldehyde prevents beta-elimination associated with the lyase activity of type I base excision repair enzymes. The interactions of the C2'-oxidized abasic site with Klenow exo(-) and repair enzymes suggest that the lesion will be mutagenic and that it will be removed by strand displacement synthesis and flap endonuclease processing via a long patch repair mechanism.